WO2006007486A2 - Compositions et procedes pour la modulation du ror$g(g)t - Google Patents

Compositions et procedes pour la modulation du ror$g(g)t Download PDF

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WO2006007486A2
WO2006007486A2 PCT/US2005/022649 US2005022649W WO2006007486A2 WO 2006007486 A2 WO2006007486 A2 WO 2006007486A2 US 2005022649 W US2005022649 W US 2005022649W WO 2006007486 A2 WO2006007486 A2 WO 2006007486A2
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cells
rorγt
gene
vaccine
cell
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PCT/US2005/022649
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WO2006007486A3 (fr
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Daniel Littman
Gerard Eberl
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New York University
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Priority to CA002572334A priority Critical patent/CA2572334A1/fr
Priority to EP05786040A priority patent/EP1771204A4/fr
Priority to JP2007519313A priority patent/JP2008505080A/ja
Publication of WO2006007486A2 publication Critical patent/WO2006007486A2/fr
Priority to US11/524,501 priority patent/US20070154487A1/en
Publication of WO2006007486A3 publication Critical patent/WO2006007486A3/fr
Priority to US14/589,336 priority patent/US20150218563A1/en
Priority to US15/358,668 priority patent/US20170159057A1/en

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    • A61K2239/50—Colon
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    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • This invention relates to novel methods and compositions for modulation of intestinal immunity.
  • the invention provides for a means of either enhancing mucosal immunity to a preselected antigen for which immunity is desired, or for diminishing the inflammation associated with intestinal disorders such as Crohn's disease, inflammatory bowel disease or H. pylori associated ulcers.
  • the gut-associated lymphoid tissue includes mesenteric lymph nodes (mLNs), Peyer's patches (PPs), the appendix and isolated lymphoid follicles (ILFs) ( H. Hamada et al., J Immunol 168, 57 (2002).) It also includes lymphocytes residing in the intestinal lamina intestinal (LPLs) and within the single layer of intestinal epithelial cells (intraepithelial lymphocytes, IELs) ( D. Guy-Grand, P. Vassalli, Curr Opin Immunol 14, 255 (2002); A. Hayday, E. Theodoridis, E. Ramsburg, J.
  • T cells present in the mLNs and PPs share the characteristics of mainstream peripheral ⁇ T cells (bearing the ⁇ T cell antigen receptor, TCR), whereas LPLs and IELs are enriched in ⁇ T cells, and most IELs uniquely express CD8 ⁇ homodimers.
  • CD8 ⁇ + , ⁇ and ⁇ IELs develop, and can be derived from bone marrow and fetal liver or intestine grafts into lymphopenic mice (B. Rocha, P. Vassalli, D. Guy-Grand, J Exp Med 180, 681 (1994); L. Lefrancois, S.
  • CPs hematopoietic CD3 " c-kit + IL- 7Ra + cells (CP cells) that express low levels of CD3 ⁇ and germline TCR transcripts, but no pre-T ⁇ chain ( K. Suzuki et al., Immunity 13, 691 (2000)) or RAG-2 ( D.
  • the retinoic acid-related orphan receptor (ROR) ⁇ t is a member of the large family of hormone nuclear receptors that include receptors for steroids, retinoids, thyroid hormones, and vitamin D3 (Mangelsdorf DJ, et al.; (1995) Cell; 83:835-839.). Nuclear receptors are potent regulators of development, cell differentiation, and organ physiology, and members of the ROR subfamily, in particular, are required for an array of developmental and physiological processes.
  • the murine Rorg gene encodes two isoforms, ROR ⁇ and ROR ⁇ t, produced probably by initiation from two distinct promoters, although differential splicing from non-coding upstream exons cannot currently be excluded.
  • ROR ⁇ mRNA is detected in many tissues including liver, lung, muscle, heart, and brain
  • ROR ⁇ t mRNA has been detected only in immature double- positive (DP) CD4+CD8+ thymocytes and in a fetal population of CD3 " CD4+CD45+ cells (He YW, Deftos ML, Ojala EW, Bevan MJ. (1998); Immunity 9:797-806; Villey I, de Chasseval R, de Villartay JP.
  • lymph nodes LNs
  • PPs Peyer's patches
  • ROR ⁇ t is exclusively expressed in lymphoid tissue inducer (LTi) cells and is required for the generation of these cells ( G. Eberl et al., Nat Immunol 5, 64 (2004)). In the adult, ROR ⁇ t is expressed in and regulates the survival of double positive (DP) CD4 + CD8 + immature thymocytes ( Z. Sun et al., Science 288, 2369 (2000)).
  • LTi lymphoid tissue inducer
  • the present invention demonstrates that in mice rendered deficient for ROR ⁇ t through breeding the Rorc( ⁇ t) GFP allele to homozygosity, intestinal lin " c-kit + IL-7R ⁇ + cells and CPs were absent, and no intestinal GFP + cells could be observed.
  • ELFs also failed to develop, as apparent by the absence of B cell clusters characteristic of these structures (Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, Nariuchi H, Ishikawa H; (1996); J. Exp. Med.
  • intestinal ⁇ T cells and CDl Ic + cells were present in normal numbers in the mutant mice, there was substantial and specific reduction in all subsets of intestinal ⁇ T cells, including CD4 " 8 " (DN), CD4 + , CD8o ⁇ + , and CD8 ⁇ + cells, as well as a reduction in B cells and IgA in the lamina intestinal and in the feces.
  • Thl7 This Th cell subset, termed Thl7, has been proposed to have pro-inflammatory functions. The results presented herein show that ROR ⁇ t is required for the development of the potentially pro-inflammatory Thl7 cells.
  • ROR ⁇ t a gene, which is expressed exclusively in fetal lymphoid tissue inducer (LTi) cells, in immature thymocytes, in intestinal lin " c- kit + IL-7R ⁇ + cells and also in Th 17 cells in the intestine. They demonstrated that ROR ⁇ t is necessary for the development of all secondary lymphoid tissue, plus intestinal cryptopatches (CPs) and isolated lymphoid follicles (ILFs), as well as for the efficient generation of ⁇ T cells.
  • LTi fetal lymphoid tissue inducer
  • CPs intestinal cryptopatches
  • IVFs isolated lymphoid follicles
  • intestinal ROR ⁇ t + cells are equivalent in the adult to fetal LTi cells, and are thus likely to induce the formation of mucosal lymphoid tissue, such as ILFs, in response to intestinal flora or to various inflammatory stimuli.
  • this invention provides for methods of enhancing or depressing immune cell activity or function by administering a modulator of ROR ⁇ t activity, that is, an agonist or an antagonist of ROR ⁇ t, respectively.
  • a modulator of ROR ⁇ t activity that is, an agonist or an antagonist of ROR ⁇ t
  • it is desirous to enhance immune cell activity and/or function such as in an individual suffering from a hyperproliferative or cancerous disease or condition, it would be desirous to administer an agonist of ROR ⁇ t.
  • a first aspect of the invention provides a method for inhibiting the formation of immune cell aggregates in the gut of a mammal, comprising administering an inhibitor or antagonist of ROR ⁇ t.
  • the aggregates comprise isolated lymphoid follicles, including colonic patches in the gut of a mammal.
  • the invention thus provides for the use of an antagonist or inhibitor of ROR ⁇ t for inhibition of formation of immune cell aggregates in an animal, preferably but not limited to the gut of the animal.
  • the cells that are inhibited are DP thymocytes, cryptopatch (CP) cells and Th-IL17 cells.
  • the cells that are inhibited are IL- 17 producing ROR ⁇ t "1" T cells.
  • the CP cells are required for the development of isolated lymphoid follicles (ILFs).
  • the method for inhibiting the formation of immune cell aggregates in the gut results in a lack of formation of lymphocyte aggregates in the lamina intestinal and in development of intraepithelial lymphocytes.
  • the method further results in a reduction in the number of ⁇ T cells, or in IL- 17 producing ROR ⁇ t + T cells.
  • the ⁇ T cells may be selected from the group consisting of CD4 " 8 " T cells, CD4+ T cells, CD8 ⁇ + T cells, CD8 ⁇ + T cells and Th-IL17 cells.
  • the reduction in ⁇ T cells or in IL- 17 producing ROR ⁇ t + T cells occurs in the intestine, and also in tissues containing lymphoid cells, such as, but not limited to lung, liver, spleen or any other lymphoid tissue or organ that may be involved in an inflammatory disease or condition.
  • a second aspect of the invention provides a method of treating inflammatory and autoimmune diseases, comprising administering a modulator of ROR ⁇ t.
  • the modulator is an inhibitor or antagonist of ROR ⁇ t.
  • the modulator is a stimulator or agonist of ROR ⁇ t.
  • the invention also provides for the use of a modulator of ROR ⁇ t, preferably an antagonist or inhibitor of ROR ⁇ t for treating inflammatory and/or autoimmune diseases or conditions in a mammal, preferably a human, although the modulator may be used to treat other domestic or non-domestic animals, including but not limited to dogs, cats, horses, cows, pigs and rodents.
  • the inflammatory or autoimmune diseases are selected from the group consisting of arthritis, diabetes, multiple sclerosis, uveitis, rheumatoid arthritis, psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, H. pylori infections and ulcers resulting from such infection, and inflammatory bowel diseases.
  • the inflammatory bowel diseases are selected from the group consisting of Crohn's disease, ulcerative colitis, sprue and food allergies.
  • the inflammatory disease or condition involves any organ or tissue containing cells in which the presence and/or expression of ROR ⁇ t has been demonstrated.
  • a third aspect of the invention provides a method of treating an infection in a mammal comprising administering a modulator of ROR ⁇ t.
  • the modulator is a stimulator or agonist of ROR ⁇ t.
  • the modulator is an inhibitor or antagonist of ROR ⁇ t.
  • the invention also provides for the use of a modulator of ROR ⁇ t for treating an infectious disease or condition in a mammal, preferably a human, although the modulator may be used to treat other domestic or non-domestic animals, including but not limited to dogs, cats, horses, cows, pigs and rodents.
  • the modulator may be an antagonist or an agonist of ROR ⁇ t.
  • the administering results in promotion of T cell development from T cell progenitors and promotion of the formation of tertiary lymphoid organs.
  • the administering results in an increase in numbers of ⁇ T cells.
  • the administering results in an increase in the number of ROR ⁇ t + T cells that produce IL- 17.
  • the ⁇ T cells are selected from the group consisting of CD4 " 8 " T cells, CD4+ T cells, CD8 ⁇ + T cells and CD8 ⁇ + T cells.
  • a fourth aspect of the invention provides a method of inducing anti-tumor immunity in a mammal comprising administering an agonist or stimulator of ROR ⁇ t.
  • methods for development of specific immunity against tumors of the gastrointestinal tract such as, but not limited to, tumors of the stomach, bowel and intestine is envisioned.
  • methods for development of specific immunity against tumors other than those that arise in the gastrointestinal tract is envisioned.
  • treatment of tumors of the lung, liver, pancreas, breast, bone and any other solid tumor or blood borne tumor is contemplated.
  • the agonist or stimulator of ROR ⁇ t may be administered alone or in conjunction with a tumor cell vaccine or in conjunction with other anti-tumor therapies known to those skilled in the art.
  • the agonist may be administered at the same time, prior to, or after the other therapies.
  • the invention also provides for the use of a modulator of ROR ⁇ t for treating a cancerous disease or condition, or for increasing anti-tumor immunity in an animal having a cancerous condition.
  • the animal is preferably a human, although the modulator may be used to treat other domestic or non-domestic animals, including but not limited to dogs, cats, horses, cows, pigs and rodents.
  • the modulator may be an antagonist or an agonist of ROR ⁇ t
  • the development of agonists that can function as adjuvants to elicit local anti-tumor immunity is envisioned.
  • the present invention provides for a means to reduce inflammation in tumors, as well as to reduce the angiogenesis and growth of the tumor that may accompany the inflammation, since inflammation is now thought to be accompanied by angiogenesis and growth of tumors.
  • the administering results in promotion of T cell development from T cell progenitors and promotion of the formation of tertiary lymphoid organs.
  • the administering results in an increase in numbers of ⁇ T cells.
  • the administering results in an increase in numbers of ROR ⁇ t + T cells that produce IL- 17.
  • the ⁇ T cells are selected from the group consisting of CD4 " 8 " T cells, CD4+ T cells, CD8 ⁇ + T cells and CD8 ⁇ + T cells.
  • a fifth aspect of the invention provides a method of increasing the number of T cells reactive to a specific antigen, comprising administering an agonist of ROR ⁇ t in conjunction with, prior to, or subsequent to the administration of the antigen.
  • a sixth aspect of the invention provides a method of increasing the immunogenicity of a vaccine candidate, wherein an increase in T cell proliferation and responsiveness by said vaccine candidate is desirable, comprising administering to a subject in conjunction with, prior to, or subsequent to said vaccine candidate, an immunogenicity promoting amount of an agonist to ROR ⁇ t.
  • the vaccine candidate is an attenuated live vaccine or a non-replicating and/or subunit vaccine, and the method results in induction of cytolytic or memory T cells specific for the vaccine candidate.
  • the vaccine is selected from the group consisting of a tumor vaccine, a viral vaccine, a bacterial vaccine, a parasitic vaccine and vaccines for other pathogenic organisms for which a long lasting immune response is necessary to provide long term protection from infection or disease.
  • the viral vaccine is selected from the group consisting of a DNA viral vaccine, an RNA viral vaccine and a retroviral viral vaccine.
  • the vaccine is a "naked DNA vaccine" whereby genetic material (e.g., nucleic acid sequences) is used as the immunizing agent.
  • genetic material e.g., nucleic acid sequences
  • the present invention relates to the introduction of exogenous or foreign DNA molecules into an individual's tissues or cells, wherein these molecules encode an exogenous protein capable of eliciting an immune response to the protein.
  • the exogenous nucleic acid sequences may be introduced alone or in the context of an expression vector wherein the sequences are operably linked to promoters and/or enhancers capable of regulating the expression of the encoded proteins.
  • a seventh aspect of the invention provides a method of increasing mucosal immunity to a preselected antigen, comprising administering to a subject in conjunction with or subsequent to said antigen, an mucosal immunity promoting amount of an agonist to ROR ⁇ t.
  • the antigen is selected from the group consisting of a bacteria, a virus, a tumor cell and any other pathogen for which increased mucosal immunity is desired.
  • An eighth aspect of the invention provides a method of treating cancers of T cell origin, comprising administering an antagonist of ROR ⁇ t.
  • the cancers may be selected from the group consisting of acute T lymphatic leukemia (T-ALL), chronic T lymphatic leukemia (T- CLL), adult T cell leukemia (ATL), non-ATL peripheral T lymphoma (PNTL), Hodgkin's, non-Hodgkin's lymphoma and other leukemias and lymphomas exhibiting a double-positive, CD4+, CD8+ phenotype.
  • T-ALL acute T lymphatic leukemia
  • T- CLL chronic T lymphatic leukemia
  • ATL adult T cell leukemia
  • PNTL non-ATL peripheral T lymphoma
  • Hodgkin's non-Hodgkin's lymphoma and other leukemias and lymphomas exhibiting a double-positive, CD4+, CD8+ phenotype.
  • a ninth aspect of the invention provides for a method of measuring or detecting the level of ROR ⁇ t in a tissue sample from a subject, whereby the presence of ROR ⁇ t in a tissue sample is indicative of the presence of, or the potential for developing, an inflammatory or autoimmune disease or other diseases or conditions characterized by an increase in inflammatory cell numbers or activity.
  • Such conditions may include inflammatory bowel diseases, rheumatoid arthritis, type I diabetes or food allergies.
  • the absence of ROR ⁇ t may be indicative of an inability to mount a proper immune response to a pathogenic organism or tumor in a subject showing the absence of ROR ⁇ t.
  • the ability to measure the presence or absence of ROR ⁇ t in an individual may aid in the ability to determine the appropriate treatment strategy for such condition.
  • the method of measuring the level of ROR ⁇ t in a subject comprises contacting a biological sample with a ligand and detecting said ligand bound to ROR ⁇ t in the sample, wherein the detection of ligand bound to ROR ⁇ t is indicative of an inflammatory condition or an autoimmune disease.
  • the ligand is an antibody, or a derivative or fragment thereof, which specifically binds to ROR ⁇ t in the sample.
  • the ability to measure ROR ⁇ t in a sample may be accomplished using a nucleotide probe specific for ROR ⁇ t. Techniques well known in the art, e.g., quantitative or semi-quantitative RT PCR or Northern blot, can be used to measure expression levels of ROR ⁇ t.
  • the tissue sample is a biopsy sample.
  • the method for determining in a biological sample the concentration of ROR ⁇ t comprises: a. contacting said sample with a ligand under conditions wherein said ligand can form a complex with ROR ⁇ t contained in the sample; and b. determining the amount of ROR ⁇ t and of ROR ⁇ t bound by said ligand by detecting the amount of complex formed, wherein said detecting is accomplished by use of a radiolabel, an enzyme, a chromophore or a fluorescent probe.
  • the method provides for screening, diagnosis or prognosis of a disease in a subject, the diseases characterized by high levels of ROR ⁇ t, wherein the diseases are selected from the group consisting of arthritis, diabetes, multiple sclerosis, uveitis, rheumatoid arthritis, psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, H. pylori infections and ulcers resulting from such infection, inflammatory bowel diseases, autoimmune diseases, and food allergies.
  • the method comprises: (I) measuring an amount of a ROR ⁇ t gene or gene product in a tissue sample derived from the subject, wherein said ROR ⁇ t gene or gene product is:
  • nucleic acid comprising a sequence hybridizable to SEQ ED NO: 1, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence
  • the method provides a diagnostic method for determining the predisposition, the onset or the presence of an inflammatory or autoimmune disease or a food allergy in a subject.
  • the method comprises detecting in the subject the existence of a change in the level of ROR ⁇ t gene or gene product, as set forth in SEQ ED NO: 1 and SEQ ED NO: 2, or detecting a polymorphism in the ROR ⁇ t gene that affects the function of the protein.
  • the method further comprises: a) obtaining a tissue biopsy from said subject; b) permeabilizing the cells in said tissue biopsy; c) incubating said tissue biopsy or cells isolated from said tissue biopsy with one of the following: v i) an antibody specific for the ROR ⁇ t gene product, or an antibody specific for the gene product of an ROR ⁇ t gene having a polymorphism that affects the function of the protein; or ii) a nucleic acid probe specific for the ROR ⁇ t gene or a nucleic acid probe that hybridizes with an ROR ⁇ t gene having a polymorphism that affects the function of the protein; d) detecting and quantitating the amount of antibody or nucleic acid probe bound; e) comparing the amount of antibody or nucleic acid probe bound in the biopsy sample in said subject to the amount of antibody or nucleic acid probe bound in a normal tissue or cellular sample; and wherein the amount of labeled antibody or nucleic acid probe bound correlates directly with the predisposition, the onset or the
  • FIG. 1 ROR ⁇ t expression in the adult mouse.
  • A ROR ⁇ t + cells in intestinal lymphoid tissues. Longitudinal sections of small intestine and colon of adult Rorc( ⁇ t) +/GFP mice were stained as indicated, as well as for GFP (green).
  • Cryptopatches (CP), small follicles (ILFs) and Peyer's patches (PP) are from the small intestine, and large follicles (ILFs) are from the colon.
  • the relative size of these different structures is compared in the first row. Magnifications are 40Ox, except for the first row and the last panel of the last row (4Ox). Sections shown are representative of at least 10 individual sections and 5 independent experiments.
  • Cells from Rorc( ⁇ t) +/GFP adult mice (blue histograms) and control Rorc( ⁇ t) +/+ mice (red lines) were analyzed by flow cytometry for expression of GFP. Cells were gated as indicated.
  • Lin " c-kit + IL-7R ⁇ + cells represented approximately 0.5% of total intestinal mononuclear cells and 0.1 to 0.2% of total PP cells. The data shown are representative of at least 10 individual mice.
  • ROR ⁇ t is required for the generation of lin " c-kit + IL-7R ⁇ + cells, CPs, and isolated lymphoid follicles (ILFs).
  • Rorc( ⁇ t) +/GFP or Rorci ⁇ tf /+ mice 10 Rorc( ⁇ tf FP/GFP , and 5 Rorc( ⁇ tf FP/GFP I Rorcf K>-Bcl-xl TG mice were analyzed by flow cytometry.
  • All groups are compared to the corresponding wild-type control (white bars). *p ⁇ 10 "2 , **p ⁇ 10 ⁇ 3 , ***p ⁇ 10 ⁇ 5 .
  • control groups (white bars), the number of ⁇ T cells may be over-estimated due to possible contamination from remaining PP cells.
  • FIG. 3 Cell-fate mapping of ROR ⁇ t + cells.
  • Rorc( ⁇ t)-Cre TG mice express Cre under control of the Rorc( ⁇ t) locus on a BAC transgene. The Cre gene was inserted into the first exon of Rorc( ⁇ t).
  • Cd4-Cre ⁇ G mice express Cre under control of a short synthetic promoter consisting (from 5' to 3') of the murine CD4 proximal enhancer, promoter, exon 1, intron 1 containing the CD4 silencer, and part of exon 2.
  • R26R mice express GFP under control of the Rosa26 locus only after Cre-mediated excision of a L ⁇ xP-flanked Stop sequence.
  • FIG. 1 Cell fate mapping of ROR ⁇ t + or CD4+ cells
  • A Cells from thymus, spleen and intestine of adult Rorc( ⁇ t)-Cre TG IR26R (blue histograms) or control R26R mouse (red lines), were analyzed by flow cytometry for the expression of GFP. Cells were gated as indicated. The data shown are representative of 3 individual experiments.
  • B Expression of CD4 by intestinal lin " ROR ⁇ t + cells. Numbers indicate the percent cells present in each quadrant. The data shown are representative of 3 individual experiments.
  • FIG. 6 Absence of mature CPs and ILFs in LT ⁇ -deficient mice. Longitudinal sections of the small intestine of adult Ltd' " Rorc( ⁇ t) +/GFP mice were stained as indicated, as well as for GFP (green). In these mice, CP rudiments were found that consisted of small clusters of ROR ⁇ t "1" cells, but that contained very few CDlIc+ dendritic cells. No ILFs were present. ROR ⁇ t + cells expressed low amounts of CD45, only apparent in these panels when the green fluorescence was removed. Magnifications are 10Ox (first two panels) and 20Ox (last two panels). Sections shown are representative of at least 10 individual sections and 3 individual mice. [0039] Figure 7.
  • ROR ⁇ t + cells in the postnatal intestinal lamina propria Longitudinal sections of the small intestine of Rorc( ⁇ t) +/GFP mice at different times after birth were stained as indicated, as well as for EGFP (green). Magnification is 4Ox. Sections shown are representative of at least 5 individual sections and 2 independent experiments.
  • FIG. 8 ROR ⁇ t + T cells in the postnatal intestinal lamina propria: Surface Staining.
  • the mouse used is heterozygous RORgt-GFP-KI.
  • Lamina limbal lymphocytes (LPLs) were isolated from small intestine and colon. Briefly, intestinal tubes were dissected out and after removal of Peyer's Patches the tubes were opened longitudinally and cut into 1.5 cm pieces. Epithelial cells and intraepithelial lymphocytes (IELs) were removed by treating with 5 mM EDTA. The pieces were then digested with 0.5 mg/ml of each of Collagenase D (Roche) and DNAse I (Sigma) as well as 0.5 U/ml Dispase (Fisher).
  • LPLs were recovered by applying the digested intestine to a Percoll gradient (80:40).
  • Percoll gradient 80:40
  • anti-mouse CD3-PerCP 145-2C11
  • anti- TCRgd-PE GL3
  • anti-TCRb-APC H57-597
  • GFP fluorescence was detected directly.
  • the mouse used is heterozygous ROR ⁇ t-GFP-KI.
  • the lamina limbal lymphocytes (LPLs) are isolated from the small intestine by the method described in the legend from Figure 8.
  • the isolated LPLs were cultured in 96 well plates for 5h (1 x 10 6 cells per well) without any stimulation.
  • the cells were surface stained with anti-mouse TCRb-APC (BD Pharmingen) and then fixed and permeabilized for intracellular cytokine staining with rat anti-mouse IL- 17-PE (BD Pharmingen).
  • the top panel shows the flow cytometry results in B6 WT controls, the second panel are the results from the ROR ⁇ t +/" mice, and panel three are the results from the ROR ⁇ t ";" mice.
  • FIG. 10 Identification of IL-17 Producing T cells from the small intestine of Rorc(ytf mf mice: Stimulation with PMA
  • the mouse is heterozygous ROR ⁇ t-GFP-KI.
  • the lamina basement lymphocytes (LPLs) are isolated from the small intestine by the method described in the legend from Figure 8.
  • the isolated LPLs were cultured in 96 well plates for 5h (1 x 10 6 cells per well) without any stimulation or with PMA/Ionomycin (50 ng/nil PMA + 200ng/ml Ionomycin) or the wells were precoated with 5 ug/ml purified anti-CD3 + anti-CD28 Abs in PBS for the CD3/CD28 stimulation.
  • the cells were first surface stained with anti-mouse CD3-PerCP (BD Pharmingen) and anti-mouse TCRb- APC (BD Pharmingen) and then fixed and permeabilized for intracellular cytokine staining with rat anti-mouse IL- 17-PE (BD Pharmingen).
  • CD3/CD28 stimulated samples was stained with rat anti-mouse IgGl-PE (BD Pharmingen).
  • DP or double positive thymocytes are immature thymocytes that express both the CD4 and CD8 receptors on their surface.
  • Isolated lymphoid follicles or "ILF” are also known as lymphoid nodules. In the colon, “isolated lymphoid follicles” are known as colon patches or "CP”.
  • Intraepithelial lymphocytes refers to T cells located in the lining of the intestine. These T cells, also referred to as “IEL” play key roles in protecting the body from invasion by harmful bacteria and viruses, minimizing immune responses to food and harmless bacteria and in promoting the repair of the intestinal lining.
  • CP cells are unique cell clusters found in the bowel wall. These small clusters of hematopoietic cells have been detected between crypts in the wall of the small intestine.
  • IBD Inflammatory bowel disease
  • Crohn's disease and ulcerative colitis are the best known forms of IBD, and both fall into the category of "idiopathic" inflammatory bowel disease because the etiology for them is unknown.
  • Pathologic findings are generally not specific, although they may suggest a particular form of EBD.
  • Active IBD is characterized by acute inflammation.
  • Chironic IBD is characterized by architectural changes of crypt distortion and scarring. Crypt abscesses (active IBD consisting of neutrophils in crypt lumens) can occur in many forms of IBD, not just ulcerative colitis.
  • Anti-tumor immunity refers to an immune response that has been generated to a specific tumor cell or to specific cancerous tissue.
  • the response may be either a B cell (antibody) response or it may be a T cell (cell-mediated) response.
  • immunogen is used herein to describe a composition typically containing a peptide or protein, or a glycolipid as an active ingredient (i.e., antigen) used for the preparation of antibodies against the peptide or protein or the glycolipid or for eliciting a T cell response.
  • immunogenic refers to the ability of an antigen to elicit an immune response, either humoral or cell mediated.
  • An "immunogenically effective amount” as used herein refers to the amount of antigen sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, as measured by standard assays known to one skilled in the art.
  • the effectiveness of an antigen as an immunogen can be measured either by proliferation assays, by cytolytic assays, such as chromium release assays to measure the ability of a T cell to lyse its specific target cell, or by measuring the levels of B cell activity by measuring the levels of circulating antibodies specific for the antigen in serum, or by measuring the number of antigen specific colony forming units in the spleen. Furthermore, the level of protection of the immune response may be measured by challenging the immunized host with the antigen-bearing pathogen.
  • the antigen to which an immune response is desired is a virus or a tumor cell
  • the level of protection induced by the "immunogenically effective amount" of the antigen is measured by detecting the level of survival after virus or tumor cell challenge of the animals.
  • mucosal immunity refers to resistance to infection across the mucous membranes. Mucosal immunity depends on immune cells and antibodies present in the linings of reproductive tract, gastrointestinal tract and other moist surfaces of the body exposed to the outside world. Thus, a person having mucosal immunity is not susceptible to the pathogenic effects of foreign microorganisms or antigenic substances as a result of antibody secretions of the mucous membranes. Mucosal epithelia in the gastrointestinal, respiratory, and reproductive tracts produce a form of IgA (IgA, secretory) that serves to protect these ports of entry into the body. Since many pathogens enter the host by way of the mucosal surfaces, a vaccine that elicits mucosal immunity would be beneficial in terms of protection from many known pathogens, such as influenza or SARS virus.
  • T cell tolerance to specific antigens can be established by administering the antigen via the oral route, thus representing a mechanism to prevent inflammation in response to commensal bacteria, food components, etc. Accordingly, there may be a potential role for ROR ⁇ t-expressing cryptopatch cells in the process of induction of oral tolerance.
  • Subunit vaccines are cell-free vaccine prepared from purified antigenic components of pathogenic microorganisms, thus carrying less risk of adverse reactions than whole-cell preparations. These vaccines are made from purified proteins or polysaccharides derived from bacteria or viruses. They include such components as toxins and cell surface molecules involved in attachment or invasion of the pathogen to the host cell. These isolated proteins act as target proteins/antigens against which an immune response may be mounted. The proteins selected for a subunit vaccine are normally displayed on the cell surface of the pathogen, such that when the subject's immune system is subsequently challenged by the pathogen, it recognizes and mounts an immune reaction to the cell surface protein and, by extension, the attached pathogen.
  • subunit vaccines are not whole infective agents, they are incapable of becoming infective. Thus, they present no risk of undesirable virulent infectivity, a significant drawback associated with other types of vaccines.
  • Subunit molecules from two or more pathogens are often mixed together to form combination vaccines.
  • the advantages to combination vaccines is that they are generally less expensive, require fewer inoculations, and, therefore, are less traumatic to the animal.
  • a "DNA vaccine” relates to the use of genetic material (e.g., nucleic acid sequences) as immunizing agents.
  • the present invention relates to the introduction of exogenous or foreign DNA molecules into an individual's tissues or cells, wherein these molecules encode an exogenous protein capable of eliciting an immune response to the protein.
  • the exogenous nucleic acid sequences may be introduced alone or in the context of an expression vector wherein the sequences are operably linked to promoters and/or enhancers capable of regulating the expression of the encoded proteins.
  • the introduction of exogenous nucleic acid sequences may be performed in the presence of a cell stimulating agent capable of enhancing the uptake or incorporation of the nucleic acid sequences into a cell.
  • exogenous nucleic acid sequences may be administered in a composition comprising a biologically compatible or pharmaceutically acceptable carrier.
  • the exogenous nucleic acid sequences may be administered by a variety of means, as described herein, and well known in the art.
  • the DNA is linked to regulatory elements necessary for expression in the cells of the individual. Regulatory elements include a promoter and a polyadenylation signal. Other elements known to skilled artisans may also be included in genetic constructs of the invention, depending on the application.
  • the present invention may be practiced using procedures known in the art, such as those described in PCT International Application Number PCT/US90/01515, wherein methods for immunizing an individual against pathogen infection by directly injecting polynucleotides into the individual's cells in a single step procedure are presented, and in U.S. patent numbers 6,635,624; 6,586,409; 6,413,942; 6,406,705; 6,383,496.
  • An "agonist” is an endogenous substance or a drug that can interact with a receptor and initiate a physiological or a pharmacological response characteristic of that receptor (contraction, relaxation, secretion, enzyme activation, etc.).
  • An agonist has a positive intrinsic activity.
  • “Intrinsic activity” is the ability of a drug (and cell) to transduce a drug-receptor binding event into a biological response.
  • an "antagonist” or “inhibitor” is a substance such as a small organic molecule or a protein or peptide or nucleic acid molecule such as an antisense nucleic acid or a small interfering RNA molecule (siRNA) or an antibody that prevents the expression and/or function of a designated molecule, such as in the matter of the present invention, the molecule is ROR ⁇ t
  • Lamina limbal growth factor is loose connective tissue in a mucosa.
  • Lamina limbal growth factor supports the delicate mucosal epithelium, allows the epithelium to move freely with respect to deeper structures, and provides for immune defense.
  • lamina propria is relatively cellular. It has been called "connective tissue with lymphatic tendencies". Because mucosal epithelium is relatively delicate and vulnerable (i.e., rather easily breached by potential invading microorganisms, compared to epidermis), laminalitis contains numerous cells with immune function to provide an effective secondary line of defense.
  • Lymphoid tissue occurs in lamina limbal growth factor (GALT), for "Gut-Associated Lymphoid Tissue".
  • GALT Gut-Associated Lymphoid Tissue.
  • the most characteristic feature of gut-associate lymphoid tissue is the presence of clusters of lymph nodules (also called lymphoid follicles), which are sites where lymphocytes congregate. At the center of each lymph nodule is a germinal center where the lymphocytes proliferate.
  • Tertiary lymphoid organs are lymphoid tissues that develop in response to inflammatory stimuli, in contrast to secondary lymphoid organs, such as lymph nodes and Peyer's patches, that develop in the fetus following a developmental program. Tertiary lymphoid tissues are commonly found in chronically inflamed tissues that are the target of autoimmunity, such as in reumathoid arthritis, thyroiditis, and type I diabetes.
  • a "small organic molecule” is an organic compound (or organic compound complexed with an inorganic compound (e.g., metal)) that has a molecular weight of less than 3 kilodaltons, and preferably less than 1.5 kilodaltons.
  • reporter gene is a nucleic acid that is readily detectable and/or encodes a gene product that is readily detectable such as green fluorescent protein (as described in U.S. Patent No. 5,625,048 issued April 29, 1997, and WO 97/26333, published July 24, 1997, the disclosures of each are hereby incorporated by reference herein in their entireties) or luciferase.
  • phrases "pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition/symptom in the host.
  • Agent refers to all materials that may be used to prepare pharmaceutical and diagnostic compositions, or that may be compounds, such as small synthetic or naturally occurring organic compounds, nucleic acids, polypeptides, antibodies, fragments, isoforms, variants, or other materials that may be used independently for such purposes, all in accordance with the present invention.
  • Treatment refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted.
  • Diagnosis refers to diagnosis, prognosis, monitoring, characterizing, selecting patients, including participants in clinical trials, and identifying patients at risk for or having a particular disorder or clinical event or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment.
  • Subject or “patient” refers to a mammal, preferably a human, in need of treatment for a condition, disorder or disease.
  • nucleic acid refers to primers, probes, and oligomer fragments to be detected, and shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites).
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • PCR polymerase chain reaction
  • U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159 is disclosed in U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159.
  • PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA.
  • a repetitive series of reaction steps involving template denaturation, primer annealing and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment (i.e, an amplicon) whose termini are defined by the 5' ends of the primers.
  • PCR is reported to be capable of producing a selective enrichment of a specific DNA sequence by a factor of 10 9 .
  • the PCR method is also described in Saiki et al., 1985, Science, 230:1350.
  • probe refers to a labeled oligonucleotide primer, which forms a duplex structure with a sequence in the target nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the target region.
  • probes are useful for identification of a target nucleic acid sequence for ROR gamma t according to the invention. Pairs of single-stranded DNA primers can be annealed to sequences within a target nucleic acid sequence or can be used to prime DNA synthesis of a target nucleic acid sequence.
  • homologous is meant a same sense nucleic acid which possesses a level of similarity with the target nucleic acid within reason and within standards known and accepted in the art.
  • the term “homologous” may be used to refer to an amplicon that exhibits a high level of nucleic acid similarity to another nucleic acid, e.g., the template cDNA.
  • enzymatic transcription has measurable and well known error rates (depending on the specific enzyme used), thus within the limits of transcriptional accuracy using the modes described herein, in that a skilled practitioner would understand that fidelity of enzymatic complementary strand synthesis is not absolute and that the amplified nucleic acid (i.e., amplicon) need not be completely identical in every nucleotide to the template nucleic acid.
  • “Complementary” is understood in its recognized meaning as identifying a nucleotide in one sequence that hybridizes (anneals) to a nucleotide in another sequence according to the rule A ⁇ T, U and C— >G (and vice versa) and thus “matches” its partner for purposes of this definition.
  • Enzymatic transcription has measurable and well known error rates (depending on the specific enzyme used), thus within the limits of transcriptional accuracy using the modes described herein, in that a skilled practitioner would understand that fidelity of enzymatic complementary strand synthesis is not absolute and that the amplicon need not be completely matched in every nucleotide to the target or template RNA.
  • Procedures using conditions of high stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65 0 C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe.
  • T lymphocytes are a subset of lymphocytes defined by their development in the thymus and expression of a T cell receptor (TCR; ⁇ or ⁇ heterodimers). T lymphocytes do not directly recognize pathogens, but MHC /peptide complexes expressed on antigen presenting cells (APC). T lymphocytes can be characterized by the expression of CD3 (part of the TCR complex) and can be subdivided into two major classes by the expression of either CD4 or CD8. CD4+ T lymphocytes recognize class II MHC/peptide complexes whereas CD8+ T lymphocytes are restricted to class I MHC/peptide complexes. T cells have receptors on their surfaces which allow it to interact with other cells and proteins.
  • TCR T cell receptor
  • APC antigen presenting cells
  • T-cell receptor is either gamma-delta or alpha-beta heterodimer. About 95% of all T-cells will express the alpha-beta TCR. The remainder express the gamma-delta TCR. In the normal development of T-cells, the gamma-delta TCR occurs first. T-cells expressing this receptor have cytotoxic capabilities and secrete recruiting lymphokines.
  • helper cells which react with peptides complexed to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells
  • cytotoxic cells which recognize peptides bound to MHC class I molecules.
  • CD4 or CD8 coreceptors which are coexpressed on immature double- positive (DP) thymocytes but are singly expressed upon maturation.
  • TCRs T cell antigen receptors
  • DP immature double- positive
  • CD4 and CD8 bind to nonpolymorphic regions of class II and class I, respectively, and signal through their association with the cytoplasmic protein-tyrosine kinase Lck.
  • Mature T cells express either CD4 or CD8 on their surface. Most helper T cells express CD4, which binds to class II major histocompatibility complex (MHC) proteins, and most cytotoxic T cells express CD8, which binds to class I MHC proteins. In the thymus, mature CD4 + CD8 " and CD4 " CD8 + T cells expressing ⁇ T-cell antigen receptors (TCR) develop from immature thymocytes through CD4 + CD8 + ⁇ TCR + intermediates.
  • MHC major histocompatibility complex
  • Gamma/delta T cells differ from alpha/beta T cells in several ways:
  • IEL are mostly CD8 ⁇ homodimers.
  • Gamma/Delta T cells like alpha/beta T cells, develop in the thymus. However, they migrate from there into body tissues, especially epithelia (e.g., intestine, skin, lining of the vagina), and don't recirculate between blood and lymph nodes. In man, gamma/delta T cells can make up to 30% of the blood T cells. They encounter antigens on the surface of the epithelial cells that surround them rather than relying on the APCs found in lymph nodes.
  • epithelia e.g., intestine, skin, lining of the vagina
  • ⁇ T cells may represent a first line of defense against invading pathogens. Their response does seem to be quicker than that of ⁇ T cells.
  • CD8 consists of two polypeptide chains, ⁇ and ⁇ , of the Ig superfamily .
  • Cell surface-expressed CD8 exists as either ⁇ heterodimers or ⁇ homodimers.
  • Thymus- derived CD8 + CTL generally express the CD8 ⁇ heterodimer , and the binding of CD8 to MHC class I is thought to strengthen the antigen-specific binding of the TCR to the peptide/MHC class I complex.
  • the CD8 ⁇ homodimer is sufficient for binding to MHC class I.
  • the CD8-alpha-alpha receptor protein appears to mediate the survival and differentiation of precursor cells into memory T cells and the homing or survival of IELs in the intestinal epithelium.
  • CP cryptopatch cells
  • ILFs contained significant numbers of CDl Ic + cells and were predominantly found in the small intestine.
  • ILFs consisted mainly of B cells, small numbers of ⁇ T cells and an activated VCAM-I + stroma, and were predominantly found in the colon.
  • Intestinal Ror ⁇ t cells expressed IL-7R ⁇ and c-kit, and IL-7R + ⁇ cells were likewise positive for ROR ⁇ t.
  • Intestinal ROR ⁇ t cells expressed both cKit and IL-7R ⁇ and all Hn " cKit + IL-7R ⁇ + cells were likewise positive for ROR ⁇ t.
  • a subpopulation of Ror ⁇ t ⁇ cells was identified in the small intestine (but not the large intestine) and the colon of Rorc( ⁇ t) +/8 ⁇ mice that produced IL-17.
  • the present invention provides the first demonstration of a molecule (ROR ⁇ t) required for development of cryptopatches and of ILFs.
  • ROR ⁇ t a molecule required for development of cryptopatches and of ILFs.
  • Previous studies on cryptopatches proposed that they are precursors for intestinal T cells thought to develop independently of the thymus.
  • the inventors' fate mapping studies shown herein clearly demonstrate that the ROR ⁇ t-expressing cells in adult intestine are not precursors for lymphocytes or other differentiated hematopoietic cells, but are instead inducers of intestinal lymphoid tissues. Additionally, they showed that ROR ⁇ t is required for the appearance of these inducer cells, and in its absence there is no organized lymphoid tissue in the gut.
  • the inventors propose that the ROR ⁇ t-dependent intestinal inducer cells respond to external cues to initiate formation of inflammatory foci, the tertiary lymphoid tissues often found at sites of autoimmune disease.
  • intestinal ⁇ T cells are derived from precursors that express ROR ⁇ t, an orphan nuclear hormone receptor detected only in immature CD4 + CD8 + thymocytes (double positive or DP thymocytes), fetal lymphoid tissue inducer (LTi) cells, and adult intestinal cryptopatch (CP) cells.
  • LTi fetal lymphoid tissue inducer
  • CP adult intestinal cryptopatch
  • One aspect of the invention provides a method of using an antibody against the ROR ⁇ t gene product, e.g.protein (or peptides derived therefrom) or nucleic acids encoding ROR ⁇ t, to diagnose a subject having or predisposed to having, a disease characterized by high levels of ROR ⁇ t, such as inflammatory diseases, autoimmune diseases or individuals suffering from food allergies.
  • an antibody against the ROR ⁇ t gene product e.g.protein (or peptides derived therefrom) or nucleic acids encoding ROR ⁇ t
  • Elevated levels of ROR ⁇ t may be found in diseases such as arthritis, diabetes, multiple sclerosis, uveitis, rheumatoid arthritis, psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, H. pylori infections and ulcers resulting from such infection, and inflammatory bowel diseases.
  • diseases such as arthritis, diabetes, multiple sclerosis, uveitis, rheumatoid arthritis, psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, H. pylori infections and ulcers resulting from such infection, and inflammatory bowel diseases.
  • one may look for a decrease in expression of the ROR ⁇ t gene after appropriate therapy for these conditions.
  • enhanced expression levels of the ROR ⁇ t gene or gene product may be desirous when one is delivering a vaccine to an individual which should then
  • the diagnostic method of the invention provides contacting a biological sample such as a biopsy sample, tissue, or cell isolated from a subject with an antibody which binds ROR ⁇ t.
  • the antibody is allowed to bind to the ROR ⁇ t antigen to form an antibody-antigen complex.
  • the ROR ⁇ t antigen includes the ROR ⁇ t protein or peptides isolated therefrom. The conditions and time required to form the antibody-antigen complex may vary and are dependent on the biological sample being tested and the method of detection being used.
  • the antibody-antigen complex is detected using any immunoassay used to detect and/or quantitate antigens [see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988) 555-612].
  • immunoassays include antibody capture assays, antigen capture assays, and two-antibody sandwich assays.
  • an antibody capture assay the antigen is attached to solid support, and labeled antibody is allowed to bind. After washing, the assay is quantitated by measuring the amount of antibody retained on the solid support.
  • an antigen capture assay the antibody is attached to a solid support, and labeled antigen is allowed to bind.
  • the unbound proteins are removed by washing, and the assay is quantitated by measuring the amount of antigen that is bound.
  • one antibody is bound to a solid support, and the antigen is allowed to bind to this first antibody.
  • the assay is quantitated by measuring the amount of a labeled second antibody that binds to the antigen.
  • These immunoassays typically rely on labeled antigens, antibodies, or secondary reagents for detection. These proteins may be labeled with radioactive compounds, enzymes, biotin, or fluorochromes. Of these, radioactive labeling may be used for almost all types of assays. Enzyme-conjugated labels are particularly useful when radioactivity must be avoided or when quick results are needed. Biotin-coupled reagents usually are detected with labeled streptavidin. Streptavidin binds tightly and quickly to biotin and may be labeled with radioisotopes or enzymes. Fluorochromes, although requiring expensive equipment for their use, provide a very sensitive method of detection.
  • the presence or absence of the antibody-antigen complex is correlated with the presence or absence in the biological sample of the ROR ⁇ t gene product.
  • a biological sample containing elevated levels of the ROR ⁇ t gene product is indicative of an inflammatory disease or an autoimmune disease or a food allergy. Examples of such diseases have been noted above.
  • the diagnostic methods of the invention may be used as part of a routine screen in subjects suspected of having such diseases or for subjects who may be predisposed to having such diseases.
  • the diagnostic method of the invention may be used alone or in combination with other well-known diagnostic methods to confirm such diseases.
  • the diagnostic method of the invention further provides that an antibody of the invention may be used to monitor the levels of ROR ⁇ t antigen in patient samples at various intervals of drug treatment to identify whether and to which degree the drug treatment is effective in restoring health.
  • ROR ⁇ t antigen levels may be monitored using an antibody of the invention in studies evaluating efficacy of drug candidates in model systems and in clinical trials.
  • ROR ⁇ t antigen levels may be monitored in biological samples of individuals treated with known or unknown therapeutic agents. This may be accomplished with cell lines in vitro or in model systems and clinical trials, depending disease being investigated. Increased total levels of ROR ⁇ t antigen in biological samples during or immediately after treatment with a drug candidate indicates that the drug candidate may actually exacerbate the disease.
  • ROR ⁇ t antigen No change in total levels of ROR ⁇ t antigen indicates that the drug candidate is ineffective in treating the disease. A lowering in total levels of ROR ⁇ t antigen indicates that the drug candidate is effective in treating the disease. This may provide valuable information at all stages of pre-clinical drug development, clinical drug trials as well as subsequent monitoring of patients undergoing drug treatment. On the other hand, in situations where enhanced immunity is desired; i.e., where an individual is being vaccinated against a pathogen or tumor, treating such individual with an agent that increases expression of ROR ⁇ t is desired. Such agonist or enhancer of ROR ⁇ t may be delivered concomitantly with the vaccine or delivered independently of the vaccine.
  • the invention involves methods to assess quantitative and qualitative aspects of ROR ⁇ t gene or gene expression.
  • the increased expression of ROR ⁇ t gene or gene product indicates a predisposition for the development of an inflammatory disease or an autoimmune disease or a food allergy.
  • enhanced expression levels of the ROR ⁇ t gene or gene product may be desirous when one is delivering a vaccine to an individual which should then lead to enhanced expression of the ROR ⁇ t gene.
  • Techniques well known in the art e.g., quantitative or semi-quantitative RT PCR or Northern blot, can be used to measure expression levels of the ROR ⁇ t gene.
  • the measurement of ROR ⁇ t gene expression levels may include measuring naturally occurring ROR ⁇ t transcripts and variants thereof as well as non-naturally occurring variants thereof.
  • the diagnosis and/or prognosis of an inflammatory disease, an autoimmune disorder, or a food allergy in a subject is preferably directed to detecting increased levels of a naturally occurring ROR ⁇ t gene product or variant thereof.
  • the invention relates to methods of diagnosing and/or predicting an inflammatory disease or an autoimmune disease or a food allergy in a subject by measuring the expression of an ROR ⁇ t gene or gene product in a subject.
  • the increased level of mRNA encoded by an ROR ⁇ t gene (e.g., SEQ ID NO: 1), as compared to a normal sample or a predetermined normal standard would indicate the presence of an inflammatory disease or an autoimmune disease or a food allergy in said subject or the increased risk of developing an inflammatory disease or an autoimmune disease or a food allergy in said subject.
  • an ROR ⁇ t gene e.g., SEQ ID NO: 1
  • the increased level of mRNA encoded for by a ROR ⁇ t gene e.g., SEQ ID NO: 1, human DNA having accession number U16997.1, or SEQ ID NO: 3, mouse DNA having accession number AF019657
  • other related gene products e.g., SEQ ID NO: 2, human protein, or SEQ ID NO: 4, mouse protein
  • RNA from a cell type or tissue known, or suspected, to express a ROR ⁇ t gene may be isolated and tested utilizing hybridization or PCR techniques as described above.
  • the isolated cells can be derived from cell culture or from a patient.
  • the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the ROR ⁇ t gene.
  • Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the ROR ⁇ t gene, including activation or suppression of ROR ⁇ t gene expression and the presence of alternatively spliced ROR ⁇ t gene transcripts.
  • a cDNA molecule is synthesized from an RNA molecule of interest by reverse transcription. AU or part of the resulting cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR or the like.
  • the nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among ROR ⁇ t gene nucleic acid reagents. The preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides.
  • the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides.
  • enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
  • RT-PCR techniques can be utilized to detect differences in ROR ⁇ t gene transcript size that may be due to normal or abnormal alternative splicing. Additionally, such techniques can be performed using standard techniques to detect quantitative differences between levels of ROR ⁇ t gene transcripts detected in normal individuals relative to those individuals having an inflammatory disease, an autoimmune disease or a food allergy or exhibiting a predisposition towards these conditions.
  • primers and/or hybridization probes can be used, such that, in the absence of such a sequence, for example, no amplification would occur.
  • Standard Northern analyses can be performed if a sufficient quantity .of the appropriate cells or tissue can be obtained.
  • the preferred length of a probe used in a Northern analysis is 9-50 nucleotides. Utilizing such techniques, quantitative as well as size related differences between ROR ⁇ t transcripts can also be detected.
  • ROR ⁇ t gene expression assays in situ, i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
  • Nucleic acid reagents such as those described herein may be used as probes and/or primers for such in situ procedures (see, e.g., Nuovo, GJ., 1992, PCR In Situ Hybridization: Protocols And Applications, Raven Press, NY).
  • Mutations or polymorphisms within a ROR ⁇ t gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell (e.g., genomic DNA) can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to those of skill in the art. For the detection of ROR ⁇ t transcripts or ROR ⁇ t gene products, any cell type or tissue in which the ROR ⁇ t gene is expressed may be utilized.
  • Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving ROR ⁇ t gene structure, including point mutations, insertions, deletions and chromosomal rearrangements.
  • assays may include, but are not limited to, direct sequencing (Wong, C. et al, 1987, Nature 330:384), single stranded conformational polymorphism analyses (SSCP; Orita, M. et al, 1989, Proc. Natl. Acad. ScL USA 86:2766), heteroduplex analysis (Keen, TJ. et al, 1991, Genomics H: 199; Perry, DJ.
  • DGGE denaturing gradient gel electrophoresis
  • Myers Myers, R.M. et al, 1985, Nucl. Acids Res. 13:3131
  • chemical mismatch cleavage Cotton, R.G. et al, 1988, Proc. Natl. Acad. ScL USA 85:4397
  • oligonucleotide hybridization Wallace, R.B. et al, 1981, Nucl. Acids Res. 9:879; Lipshutz, RJ. et al, 1995, Biotechniques 19:442).
  • Diagnostic methods for the detection of ROR ⁇ t gene nucleic acid molecules, in patient samples or other appropriate cell sources may involve the amplification of specific gene sequences, e.g., by PCR (See Mullis, K.B., 1987, U.S. Patent No. 4,683,202), followed by the analysis of the amplified molecules using techniques well known to those of skill in the art, such as, for example, those listed above. Utilizing analysis techniques such as these, the amplified sequences can be compared to those that would be expected if the nucleic acid being amplified contained only normal copies of a ROR ⁇ t gene in order to determine whether a ROR ⁇ t gene mutation exists.
  • Candidates for therapy with the agents identified by the methods described herein are patients either suffering from an inflammatory disease, an autoimmune disorder or a food allergy or are prone to development of such disorders.
  • the agents would be modulators of ROR ⁇ t, preferably inhibitors or antagonists of ROR ⁇ t.
  • treatment of these cancers with a combination of an ROR ⁇ t inhibitor (to block at the progenitor double positive stage) with chemotherapy to eliminate differentiated tumor may be effective.
  • patients in need of being vaccinated against certain pathogenic organisms, e.g. bacteria, viruses, fungi, parasites or tumors may be in need of treatment with an agent that enhances the expression of ROR ⁇ t, or with an agonist that enhances the expression and/or activity of ROR ⁇ t.
  • the invention provides methods of treatment comprising administering to a subject an effective amount of an agent that modulates the expression and/or activity of ROR ⁇ t.
  • a "modulator of ROR ⁇ t” is defined as an agent that acts as an agonist or stimulator that enhances expression and/or activity of ROR ⁇ t or an antagonist that decreases expression and/or activity of ROR ⁇ t.
  • the agent may be identified as a compound, such as a small organic molecule that acts to antagonize expression of ROR ⁇ t, or it may be a protein or polypeptide, a nucleic acid molecule such as an antisense RNA or an siRNA molecule that prevents expression of ROR ⁇ t.
  • an agent that increases expression of ROR ⁇ t such as an agonist that can be used with a vaccine candidate for various pathogenic organisms or with a tumor vaccine.
  • the agent that acts as an agonist may be identified as a compound, such as a small organic molecule that acts to stimulate expression of ROR ⁇ t, or it may be a protein or polypeptide, or a nucleic acid molecule. It is envisioned that agonists may be developed that act directly on expression and/or activity of the ROR ⁇ t protein.
  • the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
  • the subject is preferably an animal, including but not limited to animals such as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • a non-human mammal is the subject.
  • a human mammal is the subject.
  • the agents identified by the methods described herein may be formulated as pharmaceutical compositions to be used for prophylaxis or therapeutic use to treat these patients.
  • Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, or microcapsules.
  • Methods of introduction can be enteral or parenteral and include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, topical and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions comprise a therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion
  • it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, or both.
  • compositions of the invention may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved, for example, and not by way of limitation, by local infusion during surgery, by topical application, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers or co-polymers such as Elvax (see Ruan et al , 1992, Proc Natl Acad Sci USA, 89:10872-10876).
  • administration can be by direct injection by aerosol inhaler.
  • the compound in another embodiment, can be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
  • the compound can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the airways, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release (1984) supra, vol. 2, pp. 115-138).
  • Other suitable controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533.
  • Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDs 0 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • Candidate agonists and antagonists would be tested in wild type and ROR ⁇ t knockout (ko) mice, to show lack of an effect in the ko mice.
  • candidate drugs will also tested in other animals as well (rats, dogs).
  • the target would first be to human ROR ⁇ t, and then would be tested for cross-species effects in mouse (and other species).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5O .
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unaffected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a dose range for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • Plasma levels can be measured by any technique known in the art, for example, by high performance liquid chromatography.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Normal dose ranges used for particular therapeutic agents employed for specific diseases can be found in the Physicians' Desk Reference, 54 th Edition (2000).
  • Treatments may also be achieved by administering DNA encoding the agents that increase or decrease the expression of the ROR ⁇ t gene described above in an expressible genetic construction.
  • DNA encoding the agent e.g. in the event said agent is a protein or polypeptide, may be administered to the patient using techniques known in the art for delivering DNA to the cells. For example, retroviral vectors, electroporation or liposomes may be used to deliver DNA.
  • the invention includes use of any modifications or equivalents of the above agents which do not exhibit a significantly reduced or increased activity as related to ROR ⁇ t gene expression. For example, modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included. The statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent gene products being part of the invention.
  • present agents that enhance expression of ROR ⁇ t or the ROR ⁇ t genes or gene products themselves can be used as the sole active agents, or can be used in combination with other active ingredients.
  • a compound that modulates the expression of ROR ⁇ t may be used to treat immune mediated diseases associated with the presence of inflammatory cells and the inflammatory mediators produced by these cells.
  • the agent for treating an immune mediated disease or condition, whereby the immune mediated disease is an inflammatory condition would be an antagonist or inhibitor of ROR ⁇ t expression.
  • the treatment with such an antagonist may diminish the tissue damage associated with the presence of the inflammatory cells and mediators.
  • the modulators of RORgt may be particularly effective for treating inflammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohn's disease are the two major forms of idiopathic Inflammatory Bowel Disease (EBD) in humans, and are widespread and poorly understood disorders (Kirsner, J. B., et al., eds., Inflammatory Bowel Disease: 3rd ed., Lea and Febiger, Philadelphia (1988); Goldner, F. H., et al., Idiopathic Inflammatory Bowel Disease, in Stein, J. H., ed., Internal Medicine, Little Brown & Co., Boston, pp. 369-380 (1990); Cello, J.
  • IBD Ulcerative Colitis
  • Sleisenger M. H., et al.. eds., Gastrointestinal Disease: Pathophysiology Diagnosis Management, W. B. Saunders Co., Philadelphia, p. 1435 (1989)
  • Other forms of IBD include those caused by infectious agents, drugs, or the solitary rectal ulcer syndrome and collagenous colitis.
  • the diagnosis of IBD of known and unknown etiology is difficult and sometimes impossible to make (Riddell, R. H., ed., Pathology of Drug-induced and Toxic Diseases, Churchill Livingstone, New York (1982)).
  • Colitis generally refers to a more superficial mucosal disease in contrast to Crohn's disease, which presents as a deep, often transmucosal involvement and fissures (Riddell, R. H., ed., Pathology of Drug-induced and Toxic Diseases, Churchill Livingstone, New York (1982); Morrison, B. C, et al.. eds., Gastrointestinal Pathology, 2d ed., London (1979); Fenoglio-Preiser, C. M., et al., eds., Gastrointestinal Pathology: An Atlas and Text, Raven Press, New York (1989); Goldman, H., et al., Hum. Pathol.
  • Ulcerative colitis typically involves the rectum and extends proximally without intervening uninvolved areas. These uninvolved areas are usually the hallmark of Crohn's disease.
  • the histologic features of active ulcerative colitis include, beside the superficial ulcers, infiltration by inflammatory cells (e.g., mainly lymphocytes, plasma cells, variable number of neutrophils, eosinophils and mast cells) involving extensively the lamina intestinal.
  • Crypt abscesses which are aggregates of neutrophils near and invading the crypt epithelium, are generally reliable indicators of activity, while depletion of mucin in goblet cells is a less frequent finding.
  • Noncaseating granulomas may be present in gut segments from Crohn's disease, which is often also called granulomatous colitis.
  • Eliakim et al. have demonstrated enhanced production of platelet- activating factor (PAF) during active disease and inhibition by sulfasalazine and prednisolone (Eliakim, R., et al., Gastroenterology 95:1167-1172 (1988)), thus implicating PAF as a possible mediator in the disease process.
  • PAF platelet- activating factor
  • an enhanced synthesis of eicosanoids such as prostaglandins, thromboxanes and leukotrienes has been shown in both human and experimental EBD (Schumert, R., et al., Dig. Dis. Sci. 33 Suppl.:58S-64S (1988)). These products may be involved in the pathogenesis of IBD.
  • Selective inhibition of leukotrienes may be a therapeutic strategy to reduce inflammation in IBD (Schumert, R., et al., Dig. Dis. Sci. 33 Suppl.:58S-64S (1988); Goetzl, E. J., et al., Dig. Dis. Sci. 33 Suppl.:36S-40S (1988); Allgayer, H., et al., Gastroenterology 96:1290-1300 (1989 )).
  • the immunologic alterations in IBD are primarily autoimmune in nature, with colonic autoantibodies and lymphocyte-cytotoxicity directed against colonic epithelial cells.
  • the available animal models can be divided into naturally occurring and experimentally induced IBD animal models.
  • DNBS dinitrobenzene sulfonic acid
  • TNBS 2, 4, 6-trinitro- benzensulfonic acid
  • carrageenan a model of the experimental colonic lesions produced by dinitrobenzene sulfonic acid (DNBS), 2, 4, 6-trinitro- benzensulfonic acid (TNBS) and carrageenan. These models involve tissue destruction in the colon. Intrarectal administration of 5-30 mg of TNBS in 0.25 ml of 50% ethanol in the rat produces dose-dependent colonic ulcers and inflammation which are observed by gross and light microscopic examination, and by biochemical measurement of myeloperoxidase activity in the colon at 3-4 weeks (Morris, G. P., et al., Gastroenterology 96:795-803 (1989)).
  • the inflammatory infiltrate of mucosa and submucosa included polymorphonuclear leukocytes, lymphocytes, macrophages and connective tissue mast cells. Initially, massive edema and in the healing state (6-8 weeks) fibroblasts are also detected. Granulomas are also seen in 57% of rats killed at 3 weeks.
  • Carrageenan is a sulfated polygalactose (molecular weight above 100,000) widely used in the food industry and is considered safe for human use. Degraded forms of this polysaccharide (molecular weight 20,000-40,000) administered through drinking water induce ulcerative colitis in two weeks or later in experimental animals (Beekan, W. L., Experimental inflammatory bowel disease, in: Kirsner, J. B., et al., eds., Inflammatory Bowel Disease, Lea andFebiger, Philadelphia, pp. 37-49 (1988); Onderdonk, A. B., Dig. Dis. Sci. 33 Suppl.:40S-44S (1988); Benitz, K.
  • the FMLP-induced experimental colonic lesions also represent a transition between chemically and cellularly induced animal models.
  • This bacterial peptide activates and attracts neutrophils, and causes ulcers and inflammation in the rat ileum (VonRitter, C, et al., Gastroenterology 95:651-656 (1988); VonRitter, C, et al., Gastroenterology 96:811-816 (1989)).
  • This new animal model like the TNB, has not yet been extensively used.
  • Szabo proposed a new model for ulcerative colitis, which incorporates the administration of a sulfhydryl blocker, such as N-ethylmaleimide, iodoacetamide, iodoacetate or chloroacetate (U.S. patent No., 5,214,066), to the intestinal mucosa of animals. Delivery of these agents to the colon of rodents resulted in chronic ulcerative colitis.
  • a sulfhydryl blocker such as N-ethylmaleimide, iodoacetamide, iodoacetate or chloroacetate
  • MS is a multi-factorial inflammatory disease of the human central nervous system resulting in the slowing of electrical conduction along the nerve.
  • the disease is characterized by an increase in the infiltration of inflammatory cells, loss of oligodendrocytes, and increased gliosis (astrocyte hypertrophy and proliferation).
  • Myelin is the target of this cellular autoimmune inflammatory process, leading to impaired nerve conduction (for a review, see e.g. Thompson 1996, Clin. Immunother. 5, 1-11).
  • Clinical manifestations are variable, but are usually characterized by an initial relapsing-remitting course, with acute exacerbation followed by periods of clinical stability. Over time, a steady deterioration in neurological functions takes place as the disease evolves into a chronic progressive phase. This deterioration is responsible for disabling complications and side-effects, which greatly affect quality of life and increases mortality risk of affected patients. It is estimated that close to a third of a million people in the United States have MS.
  • EAE Experimental Allergic Encephalomyelitis
  • CNS central nervous system
  • Disease can be induced in susceptible strains of mice (SJL mice) by immunization with CNS myelin antigens or alternatively, disease can be passively transferred to susceptible mice using antigen stimulated CD4+ T cells (Pettinelli, J. Immunol. 127, 1981, p. 1420).
  • EAE is widely recognized as an acceptable animal model for multiple sclerosis in primates (Alvord et al. (eds.) 1984.
  • mice Experimental allergic encephalomyelitis ⁇ A useful model for multiple sclerosis. Alan R. Liss, New York).
  • Another commonly utilized experimental MS model is a viral model, whereby an MS like disease is induced by Theiler's murine encephalomyelitis virus (TMEV) (Dal Canto, M.C., and Lipton, H.L., Am. J. Path., 88:497-500 (1977)).
  • TMEV Theiler's murine encephalomyelitis virus
  • lysolecithin model is widely accepted as a model for demyelinating conditions such as MS.
  • modulators of ROR ⁇ t may be used to treat arthritis, both rheumatoid arthritis and osteoarthritis.
  • Rheumatoid arthritis is a chronic, systemic and articular inflammatory disorder which is characterized as an imbalance in the immune system that causes an overproduction of pro-inflammatory cytokines, e.g., tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 (IL-I), and a lack of anti-inflammatory cytokines, e.g. IL-10, IL-Il.
  • cytokines e.g., tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 (IL-I), and a lack of anti-inflammatory cytokines, e.g. IL-10, IL-Il.
  • RA is characterized by synovial inflammation, which progresses to cartilage destruction, bone erosion and subsequent joint deformity.
  • the primary symptoms of RA are joint inflammation, stiffness, swelling, fatigue, difficulty moving, and pain.
  • polymorphonuclear cells, macrophages, and lymphocytes are released.
  • Activated T-lymphocytes produce cytotoxins and pro- inflammatory cytokines, while macrophages stimulate the release of prostaglandins and cytotoxins.
  • Vasoactive substances histamine, kinins, and prostaglandins are released at the site of inflammation and cause edema, warmth, erythema, and pain associated with inflamed joints.
  • the pathogenesis of rheumatoid arthritis, leading to the destruction of the joints, is characterized by two phases: 1) an exudative phase involving the microcirculation of the synovial cells that allow an influx of plasma proteins and cellular elements into the joint and 2) a chronic inflammatory phase occurring in the sub-synovium and sub-chondral bone, characterized by pannus (granulation tissue) formation in the joint space, bone erosion, and cartilage destruction.
  • pannus may form adhesions and scar tissue which causes the joint deformities characteristic of rheumatoid arthritis.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Current rheumatoid arthritis treatment consists predominantly of symptomatic relief by administration of non-steroidal anti-inflammatory drugs (NSAIDs).
  • NSAID treatment is mainly effective in the early stages of rheumatoid arthritis; it is unlikely it will produce suppression of joint inflammation if the disease is present for more than one year.
  • Gold, methotrexate, immunosuppressants and corticosteroids are also used.
  • Osteoarthritis is a disorder of the movable joints characterized by deterioration and abrasion of articular cartilage, as well as by formation of new bone at the joint periphery and usually presents as pain, which worsens with exercise, or simply an X- ray that clearly shows thinning cartilage.
  • Common joints affected are the knees, hips and spine, finger, base of thumb and base of the big toe.
  • Osteoarthritis is characterized by degenerative changes in the articular cartilage (the supporting structure) and subsequent new bone formation at the articular margins.
  • osteoarthritis As osteoarthritis progresses, the surface of the articular cartilage is disrupted and wear-particles gain access to the synovial fluid which in turn stimulates phagocytosis by macrophage cells. Thus, an inflammatory response is eventually induced in osteoarthritis.
  • Common clinical symptoms of osteoarthritis include cartilaginous and bony enlargements of the finger joints and stiffness on awakening and painful movement.
  • Osteoarthritis A natural erosion of cartilage occurs with age, but excessive loads placed on joints, obesity, heredity, trauma, decreased circulation, poor bone alignment, and repetitive stress motion play a role. Osteoarthritis may also be the result of free radical damage, thought to be a major cause of many diseases, including the aging process, cancer, heart disease and degenerative diseases.
  • NSAIDs Non-steroidal anti-inflammatory drugs
  • DMARDs disease-modifying arthritic drugs
  • CIA collagen-induced arthritis model
  • CII Type II collagen
  • modulators of ROR ⁇ t may be used to treat diabetes. Modulators of ROR ⁇ t may be particularly useful in treating insulin-dependent diabetes mellitus (IDDM).
  • IDDM insulin-dependent diabetes mellitus
  • the main clinical feature of IDDM is elevated blood glucose levels (hyperglycemia).
  • the elevated blood glucose level is caused by auto ⁇ immune destruction of insulin-producing ⁇ -cells in the islets of Langerhans of the pancreas (Bach et al. 1991, Atkinson et al. 1994).
  • NOD mouse represents a model in which auto ⁇ immunity against beta-cells is the primary event in the development of IDDM. Diabetogenesis is mediated through a multi-factorial interaction between a unique MHC class II gene and multiple, unlinked, genetic loci, as in the human disease. Moreover, the NOD mouse demonstrates beautifully the critical interaction between heredity and environment, and between primary and secondary auto-immunity. Its clinical manifestation is, for example, depending on various external conditions, most importantly on the micro-organism load of the environment in which the NOD mouse is housed.
  • STZ streptozotocin
  • STZ beta-cell toxin streptozotocin
  • STZ is taken up by the pancreatic beta cell through the glucose transporter GLUT-2. This substance decomposes intracellularly, and causes damage to DNA either by alkylation or by the generation of NO.
  • DNA strand breaks leads to the activation of the abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP), which synthesizes large amounts of the (ADP-ribose) polymer, using NAD+ as a substrate.
  • PARP poly(ADP-ribose) polymerase
  • NAD+ NAD+
  • the cellular concentration of NAD+ may then decrease to very low levels, which is thought to abrogate the ability of the cell to generate sufficient energy and, finally, to lead to cell death.
  • modulators of ROR ⁇ t particularly antagonists of ROR ⁇ t may be used to downregulate the inflammatory response in many immune related diseases or conditions
  • agonists or stimulators of ROR ⁇ t may be used in situations whereby upregulation of the immune response is desirable.
  • Any organ or tissue in which a tumor may arise may respond to therapy with an agonist or stimulator of ROR ⁇ t, since the presence/expression of ROR ⁇ t is associated with certain population of lymphoid cells that may act to directly inhibit tumor cell proliferation or may act indirectly to stimulate or activate anti-tumor T or B lymphocyte responses. Accordingly, it may be possible to identify an agent that stimulates the expression of ROR ⁇ t as described herein that may be further tested in appropriate tumor models. While the agonists of ROR ⁇ t may be useful to upregulate the immune response to any tumor antigen, tumors of the intestinal tract may be of particular interest given the results of the studies described herein.
  • CRC colorectal cancer
  • Western world 1.3 million per year and over 600,000 annual deaths.
  • the great majority of CRC cases are sporadic cancers, for which it is not possible to establish a genetic disposition.
  • Effective CRC prevention in well-defined risk groups would have a significant effect on population health.
  • focus is very much on cancer prophylaxis, in acknowledgement of the fact that surgery mostly does not suffice as the only modality and that most cytotoxic regimens are ineffective against solid tumors.
  • chemoprophylaxis covers the use of pharmacologically active, non- cytotoxic agents or naturally occurring nutrients that protect against the emergence and development of clones of mutated, malignant cells.
  • Tumor cells are known to express tumor-specific antigens on the cell surface. These antigens are believed to be poorly immunogenic, largely because they represent gene products of oncogenes or other cellular genes which are normally present in the host and are therefore not clearly recognized as nonself . Although numerous investigators have tried to target immune responses against epitopes from various tumor specific antigens, none have been successful in eliciting adequate tumor immunity in vivo (Mocellin S., (2005), Front Biosci. 10:2285-305).
  • a modulator of ROR ⁇ t particularly an agonist or stimulator of ROR ⁇ t may aid in development of appropriate immune responsiveness to the tumor antigens prevalent in the cancerous condition.
  • Models for assessment of humoral and cell mediated responses to tumor antigens are well known to those skilled in the art.
  • fluorescein isothiocyanate FITC-conjugated Annexin V
  • PE phycoerythrin
  • anti-CDllc HL3
  • anti-CD8 ⁇ 53-5.8
  • anti-CD44 Ml
  • anti-CD49b DX5
  • anti-ICAM-1 3E2
  • anti-c-kit 2B8
  • anti-NKl.l PK136
  • anti-TCR ⁇ H57-597
  • allophycocyanin (APC)-conjugated anti-CD3 ⁇ 145- 2Cl 1), anti-CDllb (Ml/70), anti-CDllc (HL3), anti-B220 (RA3-6B2), anti-Gr-1 (RB6-8C5)
  • biotin-conjugated anti-CD8 ⁇ 53-6.7
  • anti-CD45.2 104
  • anti-VCAM-1 429
  • anti-TCR ⁇ GL3
  • purified anti-CD16/32 purified anti-CD16/32
  • Rabbit anti-GFP, FITC- conjugated goat anti-rabbit, Cy3-conjugated goat anti-Armenian hamster and Alexa Fluor 647-conjugated streptavidin were purchased from Molecular Probes (Eugene, OR).
  • Biotin-conjugated anti-IL-7R ⁇ mAb was purchased from eBioscience (San Diego, CA).
  • the PE-conjugated anti-mouse IL- 17 antibody was purchased from BD Pharmingen.
  • the mouse anti-CD3PerCP (145-2C11) and anti-mouse CD28 (37.51) antibodies were purchased from BD Pharmingen.
  • the hamster monoclonal antibody to murine ROR ⁇ and ROR ⁇ t was prepared at the Sloan Kettering Cancer Center monoclonal core facility. Briefly, animals were immunized with a His-tagged ROR ⁇ expressed in bacteria, and hybridoma supernatants were screened by ELISA on a MBP-ROR ⁇ fusion protein. Supernatants of positive clones were further screened for immunoblot reactivity with ROR ⁇ in extracts from ROR ⁇ -transfected 293T cells and for immunofluorescence staining of thymic sections.
  • Immunohistochemical localization of proteins was performed by incubating the slides in the presence of primary antibodies diluted in PBS, 0.1% Triton, 1% heat inactivated goat serum (HTNGS) overnight at 4°C. Then sections were rinsed with PBS, 1% HINGS, and incubated with secondary antibodies 30 min at RT, rinsed in PBS, and cover slipped using Vectashield mounting medium (Vector Laboratories).
  • Single cell suspensions were prepared from thymus, spleen and Peyer's patches.
  • Small intestinal mononuclear cells were prepared as follows. Peyer's patches were removed, the intestine was cut into pieces less than 1 mm 3 , and incubated 1 hour at 37 0 C in 15ml DMEM containing lmg/ml collagenase D (Roche Diagnostics, Mannheim, Germany). Total intestinal cells were resuspended in a 40% isotonic Percoll solution (Pharmacia, Uppsala, Sweden) and underlaid with an 80% isotonic Percoll solution. Centrifugation for 20 min at 2000 rpm yielded the mononuclear cells at the 40-80% interface.
  • RNAse A RNAse A in 100 ⁇ l STE buffer (100 mM Tris base, 100 mM NaCl and 5 mM EDTA at pH7.5). Cells were then washed, resuspended in PBS-F and analyzed.
  • Thymocytes were isolated and cultured in DMEM medium supplemented with
  • Blocs were cut with a Microm HM500 OM cryostat (Microm, Oceanside, CA) at 8 ⁇ m (tissues) thickness and sections collected onto Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA). Slides were dried 1 hour and processed for staining, or stocked at -8O 0 C. For staining, slides were first hydrated in PBS-XG, (PBS containing 0.1% triton X-100 and 1% normal goat serum, Sigma) for 5 min and blocked with 10% goat serum and 1/100 of anti-Fc receptor mAb 2.4G2 in PBS-XG for 1 hour at room temperature.
  • PBS-XG PBS containing 0.1% triton X-100 and 1% normal goat serum, Sigma
  • ROR ⁇ t The nuclear retinoic acid related orphan receptor ROR ⁇ t is necessary for the development of LNs and PPs (Sun, Z. et al, (2000) Science 288:2369; Eberl, G. et al. (2004), Nat. Immunol. 5:64). During fetal life, ROR ⁇ t is exclusively expressed in lymphoid tissue inducer (LTi) cells and is required for the generation of these cells (Eberl, G. et al. (2004), Nat. Immunol. 5:64). In the adult, ROR ⁇ t regulates the survival of double positive (DP) CD4 + CD8 + immature thymocytes (Sun, Z. et al., (2000) Science 288:2369).
  • LTi lymphoid tissue inducer
  • ROR ⁇ t is expressed in a third type of cells, namely the cryptopatch (CP) cells (Fig. IA).
  • CP cryptopatch cells
  • ROR ⁇ t "1" cells were also found in isolated lymphoid follicles (ILFs) and in the sub-epithelial dome of PPs, but not within the intestinal epithelium or in mLNs or in periaortic LNS. Most, if not all, intestinal ROR ⁇ t "1" cells expressed both c-kit and IL- 7R ⁇ , and all lin " c-kit + IL-7R ⁇ + cells expressed ROR ⁇ t (Fig. IB and 1C).
  • intestinal ⁇ T cells but not ⁇ T cells or B cells, expressed GFP (Fig. 3B and 5A).
  • intestinal lin ' c-kit + IL- 7Ra + cells did not express GFP, probably because the T cell-specific minimal CD4 enhancer/promoter is not active in these cells, even though a substantial fraction of intestinal ROR ⁇ t + cells express CD4 (Fig. 5B).
  • intestinal ⁇ T cells are derived from DP thymocytes.
  • these results shed light on the source of TCR ⁇ IEL that express CD8 ⁇ homodimers.
  • intestinal ⁇ and ⁇ T cells are not derived from intestinal ROR ⁇ t + cells, which include the lin " c-kit + IL-7R ⁇ + CP cells.
  • intestinal ⁇ T cells are derived from DP thymocytes
  • the cell fate mapping experiments do not exclude a CP-independent extrathymic origin of ⁇ IEL (T. Lin et al., Eur J Immunol 24, 1080 (1994)), since these cells are not derived from ROR ⁇ t 1" cells.
  • ⁇ IEL are present in athymic mice does not contradict our conclusions.
  • intestinal ROR ⁇ t + cells are present in LT ⁇ -deficient mice, but fail to cluster into mature CPs (Fig. 6). Together, these data suggest that intestinal ROR ⁇ t + cells are the adult equivalent of fetal LTi cells. In accordance with this hypothesis, the data presented herein show that intestinal ROR ⁇ t + cells are required for the development of CPs and ELFs in the adult intestine. The relationship between fetal LTi, the small CPs and the more elaborate ILFs will be important to elucidate. Although ROR ⁇ t + cells are continuously present in the intestinal lamina limba from the fetus to adulthood (Fig. 7), it is unclear if they represent LTi cells that persist post-natally.
  • ILFs are small and harbor a majority of CP-like lm " c-kit + cells (H. Hamada et al., J Immunol 168, 57 (2002)). Moreover, the number of ILFs is increased in dextran sulfate-induced colitis in mice (T. W. Spahn et al., Am J Pathol 161, 2273 (2002)), as well as in Crohn's disease (E. Kaiserling, Lymphology 34, 22 (2001)) and ulcerative colitis in humans (M. M. Yeung et al., Gut 47, 215 (2000)).
  • ROR ⁇ t + may thus be an attractive therapeutic target for inflammatory bowel diseases, as well as other inflammatory or autoimmune diseases or conditions.
  • l c-kit is expressed by CD3 - EL-7Ra + cells in PP anlagen and in low amounts by
  • CD3 - CD4 + cells in newborn mesenteric LNs CD3 - CD4 + cells in newborn mesenteric LNs.
  • CD4 is expressed by 50% of LTi cells and by 30-40% of intestinal ROR ⁇ t + cells.
  • LTi cells are present in LN and PP anlagen, but do not induce activation of mesenchyma; ROR ⁇ t + cells are present in the adult intestine, but do not cluster into mature cryptopatches.
  • Ulcerative colitis is induced in Sprague Dawley rats (7-8 weeks old) by anal administration of a solution in which 90 mg of trinitrobenzenesulfonic acid (TNB) is dissolved in 1.5 ml. of 20% ethanol.
  • TNB trinitrobenzenesulfonic acid
  • Certain groups of rats are treated with various doses of the ROR ⁇ t modulator and other groups are treated with a vehicle control.
  • the preferred route of administration of the ROR ⁇ t modulator is by catheter to deliver the compound directly to the colon. Most preferably, a rubber catheter such as a Nelaton catheter No. 8 is used (Rush Company, West Germany).
  • the compound is preferably introduced about 6 cm from the rectum in the rat.
  • One of skill in the art will be familiar with the use of such catheters to deliver compounds to the desired site in rats of varying ages and weights and in other experimental animals. During the experiments rats are clinically evaluated daily, and presence or absence of diarrhea is monitored.
  • the rats are sacrificed by decapitation and evaluated for severity of colonic lesions and general colonic pathology to evaluate the development of ulcerative colitis.
  • the colon is rapidly removed, opened, rinsed in saline, blotted gently, weighed and fixed in 10% formalin.
  • Standardized sections of ileum, jejunum, duodenum, stomach, liver, pancreas, kidneys and lungs are also fixed, and processed for histologic examination. Additional sections from grossly involved and uninvolved areas of colon, ileum and jejunum are frozen and subsequently homogenized for the determination of colonic myeloperoxidase activity by the method of Bradley et al. (Bradley, P.
  • the area of blood vessels labeled with deposition of monastral blue between the damaged endothelium and vascular basement membrane are measured by stereomicroscopic planimetry (Szabo, S., et al., Gastroenterology 88:228-236 (1985); Szabo, S., et al., Scand. J. Gastroenterol. 21 Suppl.:92-96 (1986)).
  • Tissue samples from colon and ileum from rats killed up to 2 days after IA or NEM are fixed in Karnovsky's fixative for electron microscopy, dehydrated in graded ethanol, embedded, cut and stained for examination by transmission electron microscopy as described (Trier, J. S., et al., Gastroenterology 92:13-22 (1987)).
  • tissue total thickness, mucosa and muscle separated in certain experiments
  • Tekmar homogenizer a Tekmar homogenizer
  • results are stored and analyzed by computer.
  • the statistical significance of differences of the group values are calculated (for parametric data) by two-tailed Student's t-test or (with parametric statistics) by the Mann- Whitney test or the Fisher- Yates Exact Probability Test.
  • lysolecithin As a second model of demyelination, intraspinal injection of lysolecithin is used. Twelve_week_old SJL/J mice are anesthetized by intraperitoneal injection of sodium pentobarbital (0.08 mg/g). Dorsal laminectomies are performed on the upper thoracic region of the spinal cord and lysolecithin (L-lysophosphatidylcholine) (Sigma, St. Louis, MO) is injected as described (Pavelko, K.D., van Engelen, B. G. & Rodriguez, M. (1998) J. Neurosci. 18, 2498_2505).
  • lysolecithin L-lysophosphatidylcholine
  • a 34 gauge needle attached to a Hamilton syringe mounted on a stereotactic micromanipulator is used to inject 1% solution of lysolecithin in sterile PBS (pH 7.4) with Evan's blue added as a marker.
  • the needle is inserted into the dorsolateral part of the spinal cord, 1 ul of lysolecithin solution is injected, and then the needle is slowly withdrawn. The wound is sutured in two layers, and mice are allowed to recover.
  • the day of lysolecithin injection is designated day 0.
  • mice Seven days after lysolecithin injection, mice are treated with the ROR ⁇ t modulator as a bolus intraperitoneal injection or intravenously. Initially a dose response study will be done to establish the most effective dose for use in this animal model. Control mice are treated with bolus intraperitoneal or intravenous injection of vehicle control. Three weeks and five weeks after the lysolecithin injection, mice are sacrificed and one mm thick sections are prepared. The araldite block showing the largest lysolecithin induced demyelination lesion is used for quantitative analysis. The total area of the lesion is quantitated using a Zeiss interactive digital analysis system. The total number of remyelinated fibers are quantitated using a Nikon microscope/computer analysis system. The data is expressed as the number of remyelinated axons/mm 2 of lesion.
  • Lysolecithin treated mice are given various doses of the ROR ⁇ t modulator on days 0, 3, 7, 10, 14, and 17 after lysolecithin injection. Animals are killed on day 21 after lysolecithin injection. PBS or vehicle controls serve as negative controls.
  • EAE Experimental allergic encephalomyelitis
  • CNS central nervous system
  • Disease can be induced in susceptible strains of mice by immunization with CNS myelin antigens or alternatively, disease can be passively transferred to susceptible mice using antigen stimulated CD4+ T cells [Pettinelli, J. Immunol. 127, 1981, p. 1420].
  • EAE is widely recognized as an acceptable animal model for multiple sclerosis in primates [Alvord et al. (eds.) 1984. Experimental allergic encephalomyelitis ⁇ A useful model for multiple sclerosis. Alan R. Liss, New York].
  • mice Female SJL/J mice (7-10 wks) are purchased from The Jackson Laboratory, housed 5 to a cage and fed standard rodent chow diet with water ad libitum. Mice are divided into groups and certain groups are treated with vehicle control (PBS), other groups are treated with various doses of the ROR ⁇ t modulator. Mice are then immunized in two sites on the flank with 150 ⁇ g of mouse PLP peptide comprising residues 139-151. PLP was administered in 200 ⁇ l of Complete Freunds adjuvant containing 2 mg/ml Mycobacteria Tuberculosis H37RA (Difco).
  • mice On the day of immunization mice are injected intravenously with 0.75 x 10 10 Bordatella pertussis bacilli (Massachusetts Public Health Laboratories, Boston, Mass.). Ten days after immunization, spleens and lymph nodes (popliteal, axillary and brachial) are harvested and the cells resuspended in RPMI-1640 containing 10% FBS (Hyclone), 5 x 10 "5 M 2-Mercaptoethanol, 100 ⁇ g/ml streptomycin and 100 U/ml penicillin. PLP was added to the cultures at 2 ⁇ g/ml. After 96 hours, the cells are harvested, washed twice and injected i.p. into naive SJL/J mice.
  • mice are observed for clinical signs of EAE and scored on a scale of 0 to 3 as follows:
  • ROR ⁇ t modulator antagonist
  • Administration may be via the intravenous route, the oral route, the intraperitoneal route or the subcutaneous route of injection.
  • the water extract and organic solvent fractions of the pharmaceutical composition of the present invention are respectively constituted in a concentration of 0.6 mg/ml and then administered for 14 days to albino rats in an amount of 1 ml per kg of body weight once a day to determine the inhibitory effect on edema. Edema is measured daily using a precision gauge, and photographs taken.
  • ROR ⁇ t modulator Studies will be done to determine whether the ROR ⁇ t modulator can effectuate increased immunity to tumor antigens. For example, studies will be done to measure the in vivo growth of tumors, for example the Hepa 1-6 tumor cells or SMCC-I colon carcinoma cells and the mortality associated with injection of these tumors to mice, when administered alone or in combination with a ROR ⁇ t modulator. [0186] To establish that immunization with tumor cells, for example, CT-hepa 1-6 cells or SMCC-I colon carcinoma cells, when administered with a ROR ⁇ t modulator can either cure established hepatomas or colon carcinoma, or prevent animals from developing tumors due to induction of an immune response, the following studies are performed. Any established animal/tumor model may be used.
  • mice are divided into groups and all are inoculated subcutaneously with live 2 x 10 6 hepa 1-6 cells or SMCC-I cells. Some groups are treated with the tumor cells plus vehicle control and some are given various doses of the ROR ⁇ t modulator at the time of injection of the tumor cells, (the ROR ⁇ t modulator may be given either orally, IP, EVI, IV or SC). The mice are monitored weekly for development of tumors. Mortality due to a large tumor burden is also monitored.
  • mice In another study, gamma-irradiated hepa 1-6 tumor cells or SMCC-I cells are used as the vaccine.
  • Three groups of ten mice per group are inoculated subcutaneously with gamma-irradiated 1 x 10 6 hepa 1-6 cells or SMCC-I cells.
  • One group is treated with a vehicle control (PBS) at the time of injection of the irradiated tumor cells, the other two groups are given the ROR ⁇ t modulator at two different doses (low and high) at the time of injection of the irradiated tumor cells.
  • PBS vehicle control
  • mice After two weeks, mice are then injected subcutaneously with 1 x 10 6 live hepa 1-6 cells. The mice are then monitored weekly for tumor growth and mortality.
  • mice are depleted of CD8+T cells by antibody treatment before or after immunization. Depletion of CD8+ T cells either before or after immunization should abrogate the ability of the cellular vaccine to elicit anti-tumor immunity in vivo.
  • the animals injected with the tumor cells alone or in conjunction with the ROR ⁇ t modulator may be sacrificed, the spleens removed and measurement of tumor specific cytolytic T cell activity measured in a standard 51Cr release assay, known to those skilled in the art.
  • Antibodies made to the tumor antigen may also be monitored by testing the serum from the animals in standard ELISA assays.

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Abstract

La présente invention concerne l'expression du RORηt dans des cellules et des tissus, et d'effet de l'expression de ce gène, d'une part sur la prolifération de cellules immunitaires spécifique, et d'autre part dans la promotion d'agrégats de cellules immunitaires. L'invention concerne en outre des procédés et des agents susceptible de faire décroître la fonction du produit génique (la protéine) ou l'expression de ce gène chez des individus souffrant d'un état inflammatoire, d'une affection auto-immune ou d'une allergie alimentaire ou de tout autre état dans lequel il est souhaitable de bloquer une réponse immunitaire. L'invention concerne également des procédés et des agents convenant au renforcement de la fonction du RORηt avec des agonistes ou l'expression de ce gène, notamment lorsqu'il est souhaitable d'augmenter l'immunité contre une cellule pathogène ou tumorale, notamment pour l'utilisation en relation avec un vaccin.
PCT/US2005/022649 2004-07-01 2005-06-24 Compositions et procedes pour la modulation du ror$g(g)t WO2006007486A2 (fr)

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US20170159057A1 (en) 2017-06-08
JP2008505080A (ja) 2008-02-21
US20150218563A1 (en) 2015-08-06
EP1771204A4 (fr) 2008-08-13
US20070154487A1 (en) 2007-07-05

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